Abstract
Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.
Highlights
Nucleosomes are the basic units of DNA compaction and the fundamental constituents of chromatin [1]
The existence of these resistant domains suggests that the accessibility of Micrococcal nuclease (MNase) to different regions of the genome is variable, and different nucleosomes may be released from chromatin at different rates
The very characteristic “nucleosome ladder” pattern observed in gel electrophoresis of DNA resolved from MNase-digested chromatin suggests that different regions of the genome may be digested by MNase at different rates: while some parts of chromatin are only broken into large fragments, other parts are reduced to mononucleosome-size fragments
Summary
Nucleosomes are the basic units of DNA compaction and the fundamental constituents of chromatin [1]. Most yeast genes and active genes in higher eukaryotes share a stereotypical nucleosome organization of their promoters: a nucleosome-depleted region (NDR) near the transcription start site (TSS), flanked by regular arrays of nucleosomes [6,7,8,9]. This stereotypical organization has been explained by the presence of barrier complexes that bind to promoters and create an energy barrier preventing nucleosome formation [10, 11], but other determinants of nucleosome positions are known, e.g., chromatin remodelers and DNA sequence [12]
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